Various separation methods have been applied to macromolecules and cell particles. Among the various processes, partition with aqueous/aqueous polymer phase systems has several desirable features; and consequently this method provides great advantage over other separation methods. Various types of new polymer phase systems have been introduced for separation of a variety of biological samples. However, high viscosity, low interfacial tension, and relatively low density difference between the two solvent phases have produced various technical problems in performing partition with the polymer phase systems. In general, these partition methods may be divided into two categories: one is the countercurrent distribution method (CCD) and the other is the countercurrent chromatography method (CCC).
CCD uses a discontinuous partition procedure consisting of the following three steps: mixing of the two solvent phases by shaking, settling them into two layers, and transfer of the mobile phase (usually the upper phase) of each partition unit to the next partition unit. Because of high viscosity and low interfacial tension between the two phases, the use of the conventional Craig apparatus becomes impractical due to the long settling times required. A substantial improvement in separation times has been achieved by introduction of a thin-layer countercurrent distribution apparatus which provides an extremely short settling distance of a few millimeters for each phase, thus reducing the settling time down to a few minutes. More recently, a fully automated centrifugal thin-layer CCD apparatus which permits both phase separation and transfer processes in a strong centrifugal force field has been designed. However, that method still requires a considerably long separation time of one theoretical plate or one operational cycle every two minutes.
CCC has also been used for partition of various biological samples under continuous elution of the mobile phase. The method generally uses a coiled column under a centrifugal force field. High speed CCC recently developed can yield an extremely high partition efficiency of one theoretical plate per second with conventional organic/aqueous two-phase solvent systems. However, when the method is applied to partition with aqueous/aqueous polymer phase systems, high viscosity of the polymer phases causes insufficient mixing of the two solvent phases in the coiled column resulting in poor peak resolution. Consequently, the method fails to produce significant improvement over the CCD method previously described. The best results have been obtained with a special type of the coil planet centrifuge called the nonsynchronous flow-through coil planet centrifuge which generates slow rotation of a coiled column under a strong centrifugal force field. However, this apparatus is extremely complex and expensive and in addition the applicable flow rate of the mobile phase is limited due to carry over of the stationary phase.